Method for preparing a biocompatible crosslinked matrix and matrix prepared thereby

ABSTRACT

Crosslinked polymer compositions comprise a first synthetic polymer containing multiple nucleophilic groups covalently bound to a second synthetic polymer containing multiple electrophilic groups. The first synthetic polymer is preferably a synthetic polypeptide or a polyethylene glycol that has been modified to contain multiple nucleophilic groups, such as primary amino (—NH 2 )or thiol (—SH) groups. The second synthetic polymer may be a hydrophilic or hydrophobic synthetic polymer which contains, or has been derivatized to contain, two or more electrophilic groups, such as succinimidyl groups. The compositions may further comprise other components, such as naturally occurring polysaccharides or proteins (such as glycosaminoglycans or collagen) and/or biologically active agents. Also disclosed are methods for using the crosslinked polymmer compositions to effect adhesion between a first surface and a second surface; to effect tissue augmentation; to prevent the formation of surgical adhesions; and to coat a surface of a synthetic implant.

CROSS REFERENCES

This application is a divisional of U.S. application Ser. No.10/364,762, filed Feb. 10, 2003, which is a continuation of U.S.application Ser. No. 09/932,536, filed Aug. 17, 2001 and issued as U.S.Pat. No. 6,534,591 on Mar. 18, 2003, which was a continuation of U.S.application Ser. No. 09/733,739, filed Dec. 8, 2000, which is acontinuation of U.S. application Ser. No. 09/302,852, filed Apr. 30,1999 and issued as U.S. Pat. No. 6,166,130 on Dec. 26, 2000, which was acontinuation of U.S. application Ser. No. 09/229,851, filed Jan. 13,1999 and issued as U.S. Pat. No. 6,051,648 on Apr. 18, 2000, which was acontinuation of U.S. application Ser. No. 08/769,806, filed Dec. 18,1996 and issued as U.S. Pat. No. 5,874,500 on Feb. 23, 1999, which was acontinuation-in-part of U.S. application Ser. No. 08/573,799, filed Dec.18, 1995, now abandoned, all of which applications are incorporatedherein by reference in full, and to which we claim priority under 35U.S.C. §120.

FIELD OF THE INVENTION

This invention relates generally to crosslinked polymer compositionscomprising a first cynthetic polymer containing multiple nucleophilicgroups crosslinked using a second synthetic polymer containing multipleeletrophilic groups, and to methods of using such compositions asbioadhesives, for tissue augmentation, in the prevention of surgicaladhesions, and for coating surfaces of synthetic implants, as drugdelivery matrices and for ophalmic applications.

BACKGROUND OF THE INVENTION

U.S. Pat. No. 5,162,430, issued Nov. 10, 1992, to Rhee et al., andcommonly owned by the assignee of the present invention, disclosescollagen—synthetic polymer conjugates prepared by covalently bindingcollagen to synthetic hydrophilic polymers such as various derivativesof polyethylene glycol.

Commonly owned U.S. Pat. No. 5,324,775, issued Jun. 28, 1994, to Rhee etal., discloses various insert, naturally occurring, biocompatiblepolymers (such as polysaccharides) covalently bound to synthetic,non-immunogenic, hydrophilic polyethylene glycol polymers.

Commonly owned U.S. Pat. No. 5,328,955, issued Jul. 12, 1994, to Rhee etal., discloses various activated forms of polyethylene glycol andvarious linkages which can be used to produce collagen—synthetic polymerconjugates having a range of physical and chemical properties.

Commonly owned, copending U.S. application Ser. No. 08/403,358, filedMar. 14, 1995, discloses a crosslinked biomaterial composition that isprepared using a hydrophobic crosslinking agent, or a mixture ofhydrophilic and hydrophobic crosslinking agents. Preferred hydrophobiccrosslinking agents include any hydrophobic polymer that contains, orcan be chemically derivatized to contain, two or more succinimidylgroups.

Commonly owned, copending U.S. application Ser. No. 08/403,360, filedMar. 14, 1995, discloses a composition useful in the prevention ofsurgical adhesions comprising a substrate material and an anti-adhesionbinding agent, where the substrate material preferably comprisescollagen and the binding agent preferably comprises at least onetissue-reactive functional group and at least one substrate-reactivefunctional group.

Commonly owned, U.S. application Ser. No. 08/476,825, filed Jun. 7,1995, by Rhee et al., discloses bioadhesive compositions comprisingcollagen crosslinked using a multifunctional activated synthetichydrophilic polymer, as well as methods of using such compositions toeffect adhesion between a first surface and a second surface, wherein atleast one of the first and second surfaces is preferably a native tissuesurface.

Japanese patent publication No. 07090241 discloses a composition usedfor temporary adhesion of a lens material to a support, to mount thematerial on a machining device, comprising a mixture of polyethyleneglycol, having an average molecular weight in the range of 1000-5000,and poly-N-vinylpyrrolidone, having an average molecular weight in therange of 30,000 -200,000.

West and Hubbell, Biomaterials (1995) 16:1153-1156, disclose theprevention of post-operative adhesions using a photopolymerizedpolyethylene glycol-co-lactic acid diacrylate hydrogel and a physicallycrosslinked polyethylene glycol-co-polypropylene glycol hydrogel,Poloxamer 407®.

Each publication cited above and herein is incorporated herein byreference in its entirety to describe and disclose the subject matterfor which it is cited.

We now disclose a detailed description of preferred embodiments of thepresent invention, including crosslinked polymer compositions comprisingsynthetic polymers which contain multiple nucleophilic groupscrosslinked using synthetic polymers containing multiple electrophilicgroups, and methods for using these compositions to effect adhesionbetween a first surface and a second surface (wherein at least one ofthe first and second surfaces is preferably a native tissue surface) orto effect the augmentation of tissue, or to prevent surgical adhesion,or to coat surfaces of synthetic implants, or for delivering drugs orother active agents, or for ophthalmic applications.

SUMMARY OF THE INVENTION

The present invention discloses a crosslinked polymer compositioncomprising a first synthetic polymer containing two or more nucleophilicgroups, and a second synthetic polymer containing two or moreelectrophilic groups which are capable of covalently bonding to oneanother to form a three dimensional matrix.

A preferred composition of the invention comprises polyethylene glycolcontaining two or more primary amino groups as the first syntheticpolymer, and polyethylene glycol containing two or more succinimidylgroups (a five-membered ring structure represented herein as —N(COCH₂)₂)as the second synthetic polymer.

In a general method for preparing a composition for the delivery of anegatively charged compound (such as a protein or drug), a firstsynthetic polymer containing two or more nucleophilic groups is reactedwith a second synthetic polymer containing two or more electrophilicgroups, wherein the first synthetic polymer is present in molar excessin comparison to the second synthetic polymer, to form a positivelycharged matrix, which is then reacted with a negatively chargedcompound. In a general method for preparing a matrix for the delivery ofa positively charged compound, a first synthetic polymer containing twoor more nucleophilic groups is reacted with a second synthetic polymercontaining two or more electrophilic groups, wherein the secondsynthetic polymer is present in molar excess in comparison to the firstsynthetic polymer, to form a negatively charged matrix, which is thenreacted with a positively charged compound.

In a general method for effecting the nonsurgical attachment of a firstsurface to a second surface, a first synthetic polymer containing two ormore nucleophilic groups is mixed with a second synthetic polymercontaining two or more electrophilic groups to provide a reactionmixture; the reaction mixture is applied to a first surface beforesubstantial crosslinking has occurred; and the first surface iscontacted with a second surface to effect adhesion between the twosurfaces.

In a general method for augmenting soft or hard tissue within the bodyof a mammalian subject, a first synthetic polymer containing two or morenucleophilic groups and a second synthetic polymer containing two ormore electrophilic groups are administered simultaneously to a tissuesite in need of augmentation and the reaction mixture is allowed tocrosslink in situ to effect augmentation of the tissue. Alternatively,the first synthetic polymer and the second synthetic polymer may bemixed immediately prior to being administered to a tissue site, suchthat the majority of the crosslinking reaction proceeds in vivo.

In a general method for preventing the formation of adhesions followingsurgery, a first synthetic polymer containing two or more nucleophilicgroups is mixed with a second synthetic polymer containing two or moreelectrophilic groups to provide a reaction mixture; the reaction mixtureis applied to tissue comprising, surrounding, or adjacent to a surgicalsite before substantial crosslinking has occurred between thenucleophilic groups and the electrophilic groups; the reaction mixtureis allowed to continue crosslinking in situ until equilibriumcrosslinking has been achieved; and the surgical site is closed byconventional methodologies.

In a general method for coating a surface of a synthetic implant, afirst synthetic polymer containing two or more nucleophilic groups ismixed with a second synthetic polymer containing two or moreelectrophilic groups to provide a reaction mixture; the reaction mixtureis applied to a surface of a synthetic implant; and the components ofthe reaction mixture are allowed to crosslink with each other on thesurface of the implant.

A feature of the invention is that the crosslinked polymer compositionsare optically clear, making the compositions and methods of theinvention particularly suited for use in ophthalmic applications inwhich optical clarity is a requirement. Furthermore, the compositions ofthe invention are comprised of biocompatible, non-immunogenic componentswhich leave no toxic, potentially inflammatory or immunogenic reactionproducts at the tissue site of administration.

Another feature of the invention is that the crosslinked polymercompositions have a high compression strength and high swellability,i.e., a composition that has been dried will swell to three times (ormore) its dried size upon rehydration, and is more “elastic.” Sincethese polymers are generally very hydrophilic, they are more easilyinjected, i.e., the crosslinked composition stays as a “cohesive mass”when injected through a fine gague (27-30 gague) needle.

Yet another feature of the invention is that nucleophilic groups on thefirst synthetic polymer may covalently bind to primary amino groups onlysine residues of collagen molecules at the tissue site ofadministration, in effect, “biologically anchoring” the composition tothe host tissue.

One feature of the invention is that the components of the compositionsare non-immunogenic and do not require a “skin test” prior to beginningtreatment, as do currently available xenogeneic collagen compositions,such as those manufactured from bovine hides.

Another feature of the invention is that, unlike collagen, thecompositions of the invention are not subject to enzymatic cleavage bymatrix metalloproteinases, such as collagenase, and are therefore notreadily degradable in vivo and, as such, are expected to have greaterlong-term persistence in vivo than prior art collagen compositions.

Still another feature is that, when the groups on each of the polymersutilized react to form an amide bond, the manufacturing of thecompositions of the present invention can be highly controlled renderingmore consistent quality of products.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 shows compression force versus displacement for disks(approximate dimensions: 5 mm thick×5 mm diameter) of crosslinkedpolymer compositions comprising tetra-amino PEG (10,000 MW) crosslinkedusing tetrafunctionally activated SE-PEG (10,000 MW), measured using theInstron Universal Tester, Model 4202, at a compression rate of 2 mm perminute.

FIG. 2 shows compression force versus displacement for disks(approximate dimensions: 5 mm thick×5 mm diameter) of crosslinkedpolymer compositions comprising tetra-amino PEG (10,000 MW) crosslinkedusing trifunctional activated SC-PEG (5,000 MW), measured using theInstron Universal Tester, Model 4202, at a compression rate of 2 mm perminute.

FIG. 3 shows the chemical structure of two commercially availablepolyethylene glycols containing multiple primary amino groups.

FIGS. 4 to 13 show the formation of various crosslinked syntheticpolymer compositions from hydrophilic polymers.

FIGS. 14 to 18 show the formation of various crosslinked syntheticpolymer compositions from hydrophobic polymers.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS OF THE INVENTION

In accordance with the present invention, crosslinked polymercompositions are prepared by reacting a first synthetic polymercontaining two or more nucleophilic groups with a second syntheticpolymer containing two or more electrophilic groups capable ofcovalently binding with the nucleophilic groups on the first syntheticpolymer.

The components of the present invention are non-immunogenic and, assuch, do not require a “skin test” prior to starting treatment, as doesxenogenic collagen. Also, unlike collagen, the compositions of theinvention are not subject to enzymatic cleavage by matrixmetalloproteinases (e.g., collagenase) and are therefore expected tohave greater long-term persistence in vivo than currently availablecollagen compositions.

The concept behind the present invention is that a synthetic polymercontaining multiple nucleophilic groups (represented below as “X”) willreact with a synthetic polymer containing multiple electrophilic groups(represented below as “Y”), resulting in a covalently bound polymernetwork, as follows:polymer-X_(m)+polymer-Y_(n)→polymer-Z-polymerwherein m≧2, n≧2,and m+n≧5;

X=—NH₂, —SH, —OH, —PH₂, —CO—NH—NH₂, etc., and can be the same ordifferent;

Y=—CO₂N(COCH₂)₂, —CO₂H, —CHO, —CHOCH₂, —N═C═O, SO₂CH═CH₂, —N(COCH)₂),—S—S—(C₅H₄N), etc., and can be the same or different; and

Z=functional group resulting from the union of a nucleophilic group (X)and an electrophilic group (Y).

As noted above, it is also contemplated by the present invention that Xand Y may be the same or different, i.e., the polymer may have twodifferent electrophilic groups, or two different nucleophilic groups,such as with glutathione.

The backbone of each polymer is preferably an alkylene oxide,particularly, ethylene oxide, propylene oxide, and mixtures thereof.Examples of difunctional alkylene oxides can be represented by:X-polymer-X Y-polymer-Ywherein X and Y are as defined above, and the term “polymer” represents—(CH₂CH₂O)_(n)— or —(CH(CH₃)CH₂O)_(n)— or—(CH₂CH₂O)_(n)—(CH(CH₃)CH₂O)_(n)—.

The required functional group X or Y is commonly coupled to the polymerbackbone by a linking group (represented below as “Q”), many of whichare known or possible. There are many ways to prepare the variousfunctionalized polymers, some of which are listed below: polymer -Q¹-X + polymer - Q²-Y → polymer - Q¹-Z-Q² - polymer - wherein Q = wholestructure = —O—(CH₂)_(n)— polymer - O—(CH₂)_(n)—X (or Y) —S—(CH₂)_(n)—polymer - S—(CH₂)_(n)—X (or Y) —NH—(CH₂)_(n)— polymer - NH—(CH₂)_(n)—X(or Y) —O₂C—NH—(CH₂)_(n)— polymer - O₂C—NH—(CH₂)_(n)—X (or Y)—O₂C—(CH₂)_(n)— polymer - O₂C—(CH₂)_(n)—X (or Y) —O₂C—CR¹H— polymer -O₂C—CRH—X (or Y) —O—R²—CO—NH— polymer - O—R—CO—NH—X (or Y)wherein n = 1-10 in each case;R¹ = H, CH₃, C₂H₅, etc.;R² = CH₂, CO—NH—CH₂ CH₂.Q¹ and Q² may be the same or different.

For example, when Q²=OCH₂CH₂ (there is no Q¹ in this case);Y=—CO₂N(COCH₂)₂; and X=—NH₂, —SH, or —OH, the resulting reactions and Zgroups would be as follows:

-   polymer-NH₂+polymer-OCH₂CH₂CO₂—N(COCH₂)₂→polymer-NH—OCH₂CH₂CO-polymer    (amide)-   polymer-SH+polymer-OCH₂CH₂CO₂—N(COCH₂)₂→polymer-S—OCH₂CH₂CO-polymer    (thioester)-   polymer-OH+polymer-OCH₂CH₂CO₂—N(COCH₂)₂→polymer-O—OCH₂CH₂CO-polymer    (ester)

An additional group, represented below as “D”, can be inserted betweenthe polymer and the linking group to increase-degradation of thecrosslinked polymer composition in vivo, for example, for use in drugdelivery applications.polymer-D-Q-X+polymer-D-Q-Y→polymer-D-Q-Z-Q-D-polymer-Some useful biodegradable groups “D” include lactide, glycolide,ε-caprolactone, poly(α-hydroxy acid), poly(amino acids),poly(anhydride), and various di- or tripeptides.Synthetic Polymers

In order to prepare the compositions of the present invention, it isfirst necessary to provide a first synthetic polymer containing two ormore nucleophilic groups, such as primary amino groups or thiol groups,and a second synthetic polymer containing two or more electrophilicgroups capable of covalently binding with the nucleophilic groups on thesecond synthetic polymer.

As used herein, the term “polymer” refers inter alia to polyalkyls,polyamino acids and polysaccharides. Additionally, for external or oraluse, the polymer may be polyacrylic acid or carbopol.

As used herein, the term “synthetic polymer” refers to polymers that arenot naturally occurring and that are produced via chemical synthesis. Assuch, naturally occurring proteins such as collagen and naturallyoccurring polysaccharides such as hyaluronic acid are specificallyexcluded. Synthetic collagen, and synthetic hyaluronic acid, and theirderivatives, are included. Synthetic polymers containing eithernucleophilic or electrophilic groups are also referred to herein as“multifunctionally activated synthetic polymers”. The term“multifunctionally activated” (or, simply, “activated”) refers tosynthetic polymers which have, or have been chemically modified to have,two or more nucleophilic or electrophilic groups which are capable ofreacting with one another (i.e., the nucleophilic groups react with theelectrophilic groups) to form covalent bonds. Types of multifunctionallyactivated synthetic polymers include difunctionally activated,tetrafunctionally activated, and star-branched polymers.

Multifunctionally activated synthetic polymers for use in the presentinvention must contain at least two, more preferably, at least three,functional groups in order to form a three-dimensional crosslinkednetwork with synthetic polymers containing multiple nucleophilic groups(i.e., “multi-nucleophilic polymers”). In other words, they must be atleast difunctionally activated, and are more preferably trifunctionallyor tetrafunctionally activated. If the first synthetic polymer is adifunctionally activated synthetic polymer, the second synthetic polymermust contain three or more functional groups in order to obtain athree-dimensional crosslinked network. Most preferably, both the firstand the second synthetic polymer contain at least three functionalgroups.

Synthetic Polymers Containing Multiple Nucleophilic Groups

Synthetic polymers containing multiple nucleophilic groups are alsoreferred to generically herein as “multi-nucleophilic polymers”. For usein the present invention, multi-nucleophilic polymers must contain atleast two, more preferably, at least three, nucleophilic groups. If asynthetic polymer containing only two nucleophilic groups is used, asynthetic polymer containing three or more electrophilic groups must beused in order to obtain a three-dimensional crosslinked network.

Preferred multi-nucleophilic polymers for use in the compositions andmethods of the present invention include synthetic polymers thatcontain, or have been modified to contain, multiple nucleophilic groupssuch as primary amino groups and thiol groups. Preferredmulti-nucleophilic polymers include: (i) synthetic polypeptides thathave been synthesized to contain two or more primary amino groups orthiol groups; and (ii) polyethylene glycols that have been modified tocontain two or more primary amino groups or thiol groups. In general,reaction of a thiol group with an electrophilic group tends to proceedmore slowly than reaction of a primary amino group with an electrophilicgroup.

Preferred multi-nucleophilic polypeptides are synthetic polypeptidesthat have been synthesized to incorporate amino acids containing primaryamino groups (such as lysine) and/or amino acids containing thiol groups(such as cysteine). Poly(lysine), a synthetically produced polymer ofthe amino acid lysine (145 MW), is particularly preferred. Poly(lysine)shave been prepared having anywhere from 6 to about 4,000 primary aminogroups, corresponding to molecular weights of about 870 to about580,000.

Poly(lysine)s for use in the present invention preferably have amolecular weight within the range of about 1,000 to about 300,000; morepreferably, within the range of about 5,000 to about 100,000; mostpreferably, within the range of about 8,000 to about 15,000.Poly(lysine)s of varying molecular weights are commercially availablefrom Peninsula Laboratories, Inc. (Belmont, Calif.).

Polyethylene glycol can be chemically modified to contain multipleprimary amino or thiol groups according to methods set forth, forexample, in Chapter 22 of Poly(ethylene Glycol) Chemistry: Biotechnicaland Biomedical Applications, J. Milton Harris, ed., Plenum Press, NY(1992). Polyethylene glycols which have been modified to contain two ormore primary amino groups are referred to herein as “multi-amino PEGs”.Polyethylene glycols which have been modified to contain two or morethiol groups are referred to herein as “multi-thiol PEGs”. As usedherein, the term “polyethylene glycol(s)” includes modified and orderivatized polyethylene glycol(s).

Various forms of multi-amino PEG are commercially available fromShearwater Polymers (Huntsville, Ala.) and from Texaco Chemical Company(Houston, Tex.) under the name “Jeffamine”. Multi-amino PEGs useful inthe present invention include Texaco's Jeffamine diamines (“D” series)and triamines (“T” series), which contain two and three primary aminogroups per molecule, respectively. General structures for the Jeffaminediamines and triamines are shown in FIG. 3.

Polyamines such as ethylenediamine (H₂N—CH₂ CH₂—NH₂),tetramethylenediamine (H₂N—(CH₂)₄—NH₂), pentamethylenediamine(cadaverine) (H₂N—(CH₂)₅—NH₂), hexamethylenediamine (H₂N—(CH₂)₆—NH₂),bis(2-hydroxyethyl)amine (HN—(CH₂CH₂OH)₂), bis(2)aminoethyl)amine(HN—(CH₂CH₂NH₂)₂), and tris(2-aminoethyl)amine (N—(CH₂CH₂NH₂)₃) may alsobe used as the synthetic polymer containing multiple nucleophilicgroups.

Synthetic Polymers Containing Multiple Electrophilic Groups

Synthetic polymers containing multiple electrophilic groups are alsoreferred to herein as “multi-electrophilic polymers.” For use in thepresent invention, the multifunctionally activated synthetic polymersmust contain at least two, more preferably, at least three,electrophilic groups in order to form a three-dimensional crosslinkednetwork with multi-nucleophilic polymers

Preferred multi-electrophilic polymers for use in the compositions ofthe invention are polymers which contain two or more succinimidyl groupscapable of forming covalent bonds with electrophilic groups on othermolecules. Succinimidyl groups are highly reactive with materialscontaining primary amino (—NH₂) groups, such as multi-amino PEG,poly(lysine), or collagen. Succinimidyl groups are slightly lessreactive with materials containing thiol (—SH) groups, such asmulti-thiol PEG or synthetic polypeptides containing multiple cysteineresidues.

As used herein, the term “containing two or more succinimidyl groups” ismeant to encompass polymers which are commercially available containingtwo or more succinimidyl groups, as well as those that must bechemically derivatized to contain two or more succinimidyl groups. Asused herein, the term “succinimidyl group” is intended to encompasssulfosuccinimidyl groups and other such variations of the “generic”succinimidyl group. The presence of the sodium sulfite moiety on thesulfosuccinimidyl group serves to increase the solubility of thepolymer.

Hydrophilic Polymers

Hydrophilic polymers and, in particular, various polyethylene glycols,are preferred for use in the compositions of the present invention. Asused herein, the term “PEG” refers to polymers having the repeatingstructure (OCH₂ CH₂)_(n).

Structures for some specific, tetrafunctionally activated forms of PEGare shown in FIGS. 4 to 13, as are generalized reaction productsobtained by reacting tetrafunctionally activated PEGs with multi-aminoPEGs. As depicted in the Figures, the succinimidyl group is afive-member ring structure represented as —N(COCH₂)₂. In FIGS. 4 to 13,the symbol {circumflex over ()} denotes an open linkage.

FIG. 4 shows the reaction of tetrafunctionally activated PEGsuccinimidyl glutarate, referred to herein as SG-PEG, with multi-aminoPEG, and the reaction product obtained thereby.

Another activated form of PEG is referred to as PEG succinimidylpropionate

(SE-PEG). The structural formula for tetrafunctionally activated SE-PEGand the reaction product obtained by reacting it with multi-amino PEGare shown in FIG. 5. In a general structural formula for the compound,the subscript 3 is replaced with an “m”. In the embodiment shown in FIG.4, m=3, in that there are three repeating CH₂ groups on either side ofthe PEG.

The structure in FIG. 5 results in a conjugate which includes an “ether”linkage which is less subject to hydrolysis. This is distinct from theconjugate shown in FIG. 4, wherein an ester linkage is provided. Theester linkage is subject to hydrolysis under physiological conditions.

Yet another functionally activated form of polyethylene glycol, whereinm=2, is shown in FIG. 6, as is the conjugate formed by reacting thetetrafunctionally activated PEG with a multi-amino PEG.

Another functionally activated PEG similar to the compounds of FIGS. 5and 6 is provided when m=1. The structural formula of thetetrafunctionally activated PEG and resulting conjugate formed byreacting the activated PEG with multi-amino PEG are shown in FIG. 7. Itis noted that this conjugate includes both an ether and a peptidelinkage. These linkages are stable under physiological conditions.

Another functionally activated form of PEG is referred to as PEGsuccinimidyl succinamide (SSA-PEG). The structural formula for thetetrafunctionally activated form of this compound and the reactionproduct obtained by reacting it with multi-amino PEG are shown in FIG.8. In the structure shown in FIG. 8, m=2; however, related compounds,wherein m=1 or m=3-10, may also be used in the compositions of theinvention.

The structure in FIG. 8 results in a conjugate which includes an “amide”linkage which, like the ether linkage previously described, is lesssubject to hydrolysis and is therefore more stable than an esterlinkage.

Yet another activated form of PEG is provided when m=0. This compound isreferred to as PEG succinimidyl carbonate (SC-PEG). The structuralformula of tetrafunctionally activated SC-PEG and the conjugate formedby reacting it with multi-amino PEG are shown in FIG. 9.

As discussed above, preferred activated polyethylene glycol derivativesfor use in the invention contain succinimidyl groups as the reactivegroup. However, different activating groups can be attached at sitesalong the length of the PEG molecule. For example, PEG can bederivatized to form functionally activated PEG propion aldehyde (A-PEG),the tetrafunctionally activated form of which is shown in FIG. 10, as isthe conjugate formed by the reaction of A-PEG with multi-amino PEG. Thelinkage shown in FIG. 10 is referred to as a —(CH₂)_(m)—NH— linkage,where m=1-10.

Yet another form of activated polyethylene glycol is functionallyactivated PEG glycidyl ether (E-PEG), of which the tetrafunctionallyactivated compound is shown in FIG. 11, as is the conjugate formed byreacting such with multi-amino PEG.

Another activated derivative of polyethylene glycol is functionallyactivated PEG-isocyanate (I-PEG), which is shown in FIG. 12, along withthe conjugate formed by reacting such with multi-amino PEG.

Another activated derivative of polyethylene glycol is functionallyactivated PEG-vinylsulfone (V-PEG), which is shown in FIG. 13, below,along with the conjugate formed by reacting such with multi-amino PEG.

Preferred multifunctionally activated polyethylene glycols for use inthe compositions of the present invention are polyethylene glycolscontaining succinimidyl groups, such as

SG-PEG and SE-PEG (shown in FIGS. 4-7), preferably in trifunctionally ortetrafunctionally activated form.

Many of the activated forms of polyethylene glycol described above arenow available commercially from Shearwater Polymers, Huntsville, Ala.,and Union Carbide, South Charleston, W. Va.

Hydrophobic Polymers

Hydrophobic polymers can also be used to prepare the compositions of thepresent invention. Hydrophobic polymers for use in the present inventionpreferably contain, or can be derivatized to contain, two or moreelectrophilic groups, such as succinimidyl groups, most preferably, two,three, or four electrophilic groups. As used herein, the term“hydrophobic polymer” refers to polymers which contain a relativelysmall proportion of oxygen or nitrogen atoms.

Hydrophobic polymers which already contain two or more succinimidylgroups include, without limitation, disuccinimidyl suberate (DSS),bis(sulfosuccinimidyl) suberate (BS³), dithiobis(succinimidylpropionate)(DSP), bis(2-succinimidooxycarbonyloxy) ethyl sulfone (BSOCOES), and3,3′-dithiobis(sulfosuccinimidylpropionate (DTSPP), and their analogsand derivatives. The above-referenced polymers are commerciallyavailable from Pierce (Rockford, Ill.), under catalog Nos. 21555, 21579,22585, 21554, and 21577, respectively. Structural formulas for theabove-referenced polymers, as well as generalized reaction productsobtained by reacting each of these polymers with amino PEG, are shownbelow in FIGS. 14-18, respectively.

Preferred hydrophobic polymers for use in the invention generally have acarbon chain that is no longer than about 14 carbons. Polymers havingcarbon chains substantially longer than 14 carbons generally havevery-poor solubility in aqueous solutions and, as such, have very longreaction times when mixed with aqueous solutions of synthetic polymerscontaining multiple nucleophilic groups.

Derivatization of Polymers to Contain Functional Groups

Certain polymers, such as polyacids, can be derivatized to contain twoor more functional groups, such as succinimidyl groups. Polyacids foruse in the present invention include, without limitation,trimethylolpropane-based tricarboxylic acid, di(trimethylolpropane)-based tetracarboxylic acid, heptanedioic acid, octanedioic acid(suberic acid), and hexadecanedioic acid (thapsic acid). Many of thesepolyacids are commercially available from DuPont Chemical Company.

According to a general method, polyacids can be chemically derivatizedto contain two or more succinimidyl groups by reaction with anappropriate molar amount of

N-hydroxysuccinimide (NHS) in the presence ofN,N′-dicyclohexylcarbodiimide (DCC).

Polyalcohols such as trimethylolpropane and di(trimethylol propane) canbe converted to carboxylic acid form using various methods, then furtherderivatized by reaction with NHS in the presence of DCC to producetrifunctionally and tetrafunctionally activated polymers, respectively,as described in commonly owned, copending U.S. application Ser. No.08/403,358. Polyacids such as heptanedioic acid (HOOC—(CH₂)₅—COOH),octanedioic acid (HOOC—(CH₂)₆—COOH), and hexadecanedioic acid(HOOC—(CH₂)₁₄—COOH) are derivatized by the addition of succinimidylgroups to produce difunctionally activated polymers.

Polyamines such as ethylenediamine (H₂N—CH₂ CH₂—NH₂),tetramethylenediamine (H₂N—(CH₂)₄—NH₂), pentamethylenediamine(cadaverine) (H₂N—(CH₂)₅—NH₂), hexamethylenediamine (H₂N—(CH₂)₆—NH₂),bis(2-hydroxyethyl)amine (HN—(CH₂CH₂OH)₂), bis(2)aminoethyl)amine(HN—(CH₂CH₂NH₂)₂), and tris(2-aminoethyl)amine (N—(CH₂CH₂NH₂)₃) can bechemically derivatized to polyacids, which can then be derivatized tocontain two or more succinimidyl groups by reacting with the appropriatemolar amounts of N-hydroxysuccinimide in the presence of DCC, asdescribed in U.S. application Ser. No. 08/403,358. Many of thesepolyamines are commercially available from DuPont Chemical Company.

Preparation of Crosslinked Polymer Compositions

In a general method for preparing the crosslinked polymer compositionsof the invention, a first synthetic polymer containing multiplenucleophilic groups is mixed with a second synthetic polymer containingmultiple electrophilic groups. Formation of a three-dimensionalcrosslinked network occurs as a result of the reaction between thenucleophilic groups on the first synthetic polymer and the electrophilicgroups on the second synthetic polymer.

Hereinafter, the term “first synthetic polymer” will be used to refer toa synthetic polymer containing two or more nucleophilic groups, and theterm “second synthetic polymer” will be used to refer to a syntheticpolymer containing two or more electrophilic groups. The concentrationsof the first synthetic polymer and the second synthetic polymer used toprepare the compositions of the present invention will vary dependingupon a number of factors, including the types and molecular weights ofthe particular synthetic polymers used and the desired end useapplication.

In general, we have found that when using multi-amino PEG as the firstsynthetic polymer, it is preferably used at a concentration in the rangeof about 0.5 to about 20 percent by weight of the final composition,while the second synthetic polymer is used at a concentration in therange of about 0.5 to about 20 percent by weight of the finalcomposition. For example, a final composition having a total weight of 1gram (1000 milligrams) would contain between about 5 to about 200milligrams of multi-amino PEG, and between about 5 to about 200milligrams of the second synthetic polymer.

Use of higher concentrations of both first and second synthetic polymerswill result in the formation of a more tightly crosslinked network,producing a stiffer, more robust gel. As such, compositions intended foruse in tissue augmentation will generally employ concentrations of firstand second synthetic polymer that fall toward the higher end of thepreferred concentration range. Compositions intended for use asbioadhesives or in adhesion prevention do not need to be as firm and maytherefore contain lower polymer concentrations.

Because polymers containing multiple electrophilic groups will alsoreact with water, the second synthetic polymer is generally stored andused in sterile, dry form to prevent the loss of crosslinking abilitydue to hydrolysis which typically occurs upon exposure of suchelectrophilic groups to aqueous media. Processes for preparing synthetichydrophilic polymers containing multiple electrophylic groups insterile, dry form are set forth in commonly assigned, copending U.S.application Ser. No. 08/497,573, filed Jun. 30, 1995. For example, thedry synthetic polymer may be compression molded into a thin sheet ormembrane, which can then be sterilized using gamma or, preferably,e-beam irradiation. The resulting dry membrane or sheet can be cut tothe desired size or chopped into smaller size particulates.

Polymers containing multiple nucleophilic groups are generally notwater-reactive and can therefore be stored in aqueous solution.

The crosslinked polymer compositions can also be prepared to containvarious imaging agents such as iodine or barium sulfate, or fluorine, inorder to aid visualization of the compositions after administration viaX-ray, or ¹⁹F-MRI, respectively.

Incorporation of other Components into the Crosslinked Synthetic Polymer

Naturally occurring proteins, such as collagen, and derivatives ofvarious naturally occurring polysaccharides, such as glycosaminoglycans,can additionally be incorporated into the compositions of the invention.When these other components also contain functional groups which willreact with the functional groups on the synthetic polymers, theirpresence during mixing and/or crosslinking of the first and secondsynthetic polymer will result in formation of a crosslinked syntheticpolymer-naturally occurring polymer matrix. In particular, when thenaturally occurring polymer (protein or polysaccharide) also containsnucleophilic groups such as primary amino groups, the electrophilicgroups on the second synthetic polymer will react with the primary aminogroups on these components, as well as the nucleophilic groups on thefirst synthetic polymer, to cause these other components to become partof the polymer matrix.

In general, glycosaminoglycans must be chemically derivatized bydeacetylation, desulfation, or both in order to contain primary aminogroups available for reaction with electrophilic groups on syntheticpolymer molecules. Glycosaminoglycans that can be derivatized accordingto either or both of the aforementioned methods include the following:hyaluronic acid, chondroitin sulfate A, chondroitin sulfate B (dermatansulfate), chondroitin sulfate C, chitin (can be derivatized tochitosan), keratan sulfate, keratosulfate, and heparin. Derivatizationof glycosaminoglycans by deacetylation and/or desulfation and covalentbinding of the resulting glycosaminoglycan derivatives with synthetichydrophilic polymers is described in further detail in commonlyassigned, allowed U.S. patent application Ser. No. 08/146,843, filedNov. 3, 1993.

Similarly, electrophilic groups on the second synthetic polymer willreact with primary amino groups on lysine residues or thiol groups oncysteine residues of certain naturally occurring proteins. Lysine-richproteins such as collagen and its derivatives are especially reactivewith electrophilic groups on synthetic polymers. As used herein, theterm “collagen” is intended to encompass collagen of any type, from anysource, including, but not limited to, collagen extracted from tissue orproduced recombinantly, collagen analogues, collagen derivatives,modified collagens, and denatured collagens such as gelatin. Covalentbinding of collagen to synthetic hydrophilic polymers is described indetail in commonly assigned U.S. Pat. No. 5,162,430, issued Nov. 10,1992, to Rhee et al.

In general, collagen from any source may be used in the compositions ofthe invention; for example, collagen may be extracted and purified fromhuman or other mammalian source, such as bovine or porcine corium andhuman placenta, or may be recombinantly or otherwise produced. Thepreparation of purified, substantially non-antigenic collagen insolution from bovine skin is well known in the art. Commonly owned, U.S.Pat. No. 5,428,022, issued Jun. 27, 1995, to Palefsky et al., disclosesmethods of extracting and purifying collagen from the human placenta.Commonly owned, copending U.S. application Ser. No. 08/183,648, filedJan. 18, 1994, discloses methods of producing recombinant human collagenin the milk of transgenic animals, including transgenic cows. The term“collagen” or “collagen material” as used herein refers to all forms ofcollagen, including those which have been processed or otherwisemodified.

Collagen of any type, including, but not limited to, types I, II, III,IV, or any combination thereof, may be used in the compositions of theinvention, although type I is generally preferred. Either atelopeptideor telopeptide-containing collagen may be used; however, when collagenfrom a xenogeneic source, such as bovine collagen, is used, atelopeptidecollagen is generally preferred, because of its reduced immunogenicitycompared to telopeptide-containing collagen.

Collagen that has not been previously crosslinked by methods such asheat, irradiation, or chemical crosslinking agents is preferred for usein the compositions of the invention, although previously crosslinkedcollagen may be used. Non-crosslinked atelopeptide fibrillar collagen iscommercially available from Collagen Corporation (Palo Alto, Calif.) atcollagen concentrations of 35 mg/ml and 65 mg/ml under the trademarksZyderm® I Collagen and Zyderm II Collagen, respectively. Glutaraldehydecrosslinked atelopeptide fibrillar collagen is commercially availablefrom Collagen Corporation at a collagen concentration of 35 mg/ml underthe trademark Zyplast® Collagen.

Collagens for use in the present invention are generally in aqueoussuspension at a concentration between about 20 mg/ml to about 120 mg/ml;preferably, between about 30 mg/ml to about 90 mg/ml.

Although intact collagen is preferred, denatured collagen, commonlyknown as gelatin, can also be used in the compositions of the invention.Gelatin may have the added benefit of being degradable faster thancollagen.

Because of its tacky consistency, nonfibrillar collagen is generallypreferred for use in compositions of the invention that are intended foruse as bioadhesives. The term “nonfibrillar collagen” refers to anymodified or unmodified collagen material that is in substantiallynonfibrillar form at pH 7, as indicated by optical clarity of an aqueoussuspension of the collagen.

Collagen that is already in nonfibrillar form may be used in thecompositions of the invention. As used herein, the term “nonfibrillarcollagen” is intended to encompass collagen types that are nonfibrillarin native form, as well as collagens that have been chemically modifiedsuch that they are in nonfibrillar form at or around neutral pH.Collagen types that are nonfibrillar (or microfibrillar) in native forminclude types IV, VI, and VII.

Chemically modified collagens that are in nonfibrillar form at neutralpH include succinylated collagen and methylated collagen, both of whichcan be prepared according to the methods described in U.S. Pat. No.4,164,559, issued Aug. 14, 1979, to Miyata et al., which is herebyincorporated by reference in its entirety. Due to its inherenttackiness, methylated collagen is particularly preferred for use inbioadhesive compositions, as disclosed in commonly owned, U.S.application Ser. No. 08/476,825.

Collagens for use in the crosslinked polymer compositions of the presentinvention may start out in fibrillar form, then be rendered nonfibrillarby the addition of one or more fiber disassembly agent. The fiberdisassembly agent must be present in an amount sufficient to render thecollagen substantially nonfibrillar at pH 7, as described above. Fiberdisassembly agents for use in the present invention include, withoutlimitation, various biocompatible alcohols, amino acids, inorganicsalts, and carbohydrates, with biocompatible alcohols being-particularlypreferred. Preferred biocompatible alcohols include glycerol andpropylene glycol. Non-biocompatible alcohols, such as ethanol, methanol,and isopropanol, are not preferred for use in the present invention, dueto their potentially deleterious effects on the body of the patientreceiving them. Preferred amino acids include arginine. Preferredinorganic salts include sodium chloride and potassium chloride. Althoughcarbohydrates, such as various sugars including sucrose, may be used inthe practice of the present invention, they are not as preferred asother types of fiber disassembly agents because they can have cytotoxiceffects in vivo.

Because it is opaque and less tacky than nonfibillar collagen, fibrillarcollagen is less preferred for use in bioadhesive compositions. However,as disclosed in commonly owned, U.S. application Ser. No. 08/476,825,fibrillar collagen, or mixtures of nonfibrillar and fibrillar collagen,may be preferred for use in adhesive compositions intended for long-termpersistence in vivo, if optical clarity is not a requirement.

For compositions intended for use in tissue augmentation, fibrillarcollagen is preferred because it tends to form stronger crosslinked gelshaving greater long-term persistency in vivo than those prepared usingnonfibrillar collagen.

In general, the collagen is added to the first synthetic polymer, thenthe collagen and first synthetic polymer are mixed thoroughly to achievea homogeneous composition. The second synthetic polymer is then addedand mixed into the collagen/first synthetic polymer mixture, where itwill covalently bind to primary amino groups or thiol groups on thefirst synthetic polymer and primary amino groups on the collagen,resulting in the formation of a homogeneous crosslinked network. Variousdeacetylated and/or desulfated glycosaminoglycan derivatives can beincorporated into the composition in a similar manner as that describedabove for collagen.

For use in tissue adhesion as discussed below, it may also be desirableto incorporate proteins such as albumin, fibrin or fibrinogen into thecrosslinked polymer composition to promote cellular adhesion.

In addition, the introduction of hydrocolloids such ascarboxymethylcellulose may promote tissue adhesion and/or swellability.

Administration of the Crosslinked Synthetic Polymer Compositions

The compositions of the present invention may be administered before,during or after crosslinking of the first and second synthetic polymer.Certain uses, which are discussed in greater detail below, such astissue augmentation, may require the compositions to be crosslinkedbefore administration, whereas other applications, such as tissueadhesion, require the compositions to be administered beforecrosslinking has reached “equilibrium.” The point at which crosslinkinghas reached equilibrium is defined herein as the point at which thecomposition no longer feels tacky or sticky to the touch.

In order to administer the composition prior to crosslinking, the firstsynthetic polymer and second synthetic polymer may be contained withinseparate barrels of a dual-compartment syringe. In this case, the twosynthetic polymers do not actually mix until the point at which the twopolymers are extruded from the tip of the syringe needle into thepatient's tissue. This allows the vast majority of the crosslinkingreaction to occur in situ, avoiding the problem of needle blockage whichcommonly occurs if the two synthetic polymers are mixed too early andcrosslinking between the two components is already too advanced prior todelivery from the syringe needle. The use of a dual-compartment syringe,as described above, allows for the use of smaller diameter needles,which is advantageous when performing soft tissue augmentation indelicate facial tissue, such as that surrounding the eyes.

Alternatively, the first synthetic polymer and second synthetic polymermay be mixed according to the methods described above prior to deliveryto the tissue site, then injected to the desired tissue site immediately(preferably, within about 60 seconds) following mixing.

In another embodiment of the invention, the first synthetic polymer andsecond synthetic polymer are mixed, then extruded and allowed tocrosslink into a sheet or other solid form. The crosslinked solid isthen dehydrated to remove substantially all unbound water. The resultingdried solid may be ground or comminuted into particulates, thensuspended in a nonaqueous fluid carrier, including, without limitation,hyaluronic acid, dextran sulfate, dextran, succinylated noncrosslinkedcollagen, methylated noncrosslinked collagen, glycogen, glycerol,dextrose, maltose, triglycerides of fatty acids (such as corn oil,soybean oil, and sesame oil), and egg yolk phospholipid. The suspensionof particulates can be injected through a small-gauge needle to a tissuesite. Once inside the tissue, the crosslinked polymer particulates willrehydrate and swell in size at least five-fold.

Use of Crosslinked Synthetic Polymers to Deliver Charged Compounds

By varying the relative molar amounts of the first synthetic polymer andthe second synthetic polymer, it is possible to alter the net charge ofthe resulting crosslinked polymer composition, in order to prepare amatrix for the delivery of a charged compound (such as a protein ordrug). As such, the delivery of charged proteins or drugs, which wouldnormally diffuse rapidly out of a neutral carrier matrix, can becontrolled.

For example, if a molar excess of a first synthetic polymer containingmultiple nucleophilic groups is used, the resulting matrix has a netpositive charge and can be used to ionically bind and deliver negativelycharged compounds. Examples of negatively charged compounds that can bedelivered from these matrices include various drugs, cells, proteins,and polysaccharides. Negatively charged collagens, such as succinylatedcollagen, and glycosaminoglycan derivatives, such as sodium hyaluronate,keratan sulfate, keratosulfate, sodium chondroitin sulfate A, sodiumdermatan sulfate B, sodium chondroitin sulfate C, heparin, esterifiedchondroitin sulfate C, and esterified heparin, can be effectivelyincorporated into the crosslinked polymer matrix as described above.

If a molar excess of a second synthetic polymer containing multipleelectrophilic groups is used, the resulting matrix has a net negativecharge and can be used to ionically bind and deliver positively chargedcompounds. Examples of positively charged compounds that can bedelivered from these matrices include various drugs, cells, proteins,and polysaccharides. Positively charged collagens, such as methylatedcollagen, and glycosaminoglycan derivatives, such as esterifieddeacetylated hyaluronic acid, esterified deacetylated desulfatedchondroitin sulfate A, esterified deacetylated desulfated chondroitinsulfate C, deacetylated desulfated keratan sulfate, deacetylateddesulfated keratosulfate, esterified desulfated heparin, and chitosan,can be effectively incorporated into the crosslinked polymer matrix asdescribed above.

Use of Crosslinked Synthetic Polymers to Deliver Biologically ActiveAgents

The crosslinked polymer compositions of the present invention may alsobe used for localized delivery of various drugs and other biologicallyactive agents. Biologically active agents such as growth factors may bedelivered from the composition to a local tissue site in order tofacilitate tissue healing and regeneration.

The term “biologically active agent” or “active agent” as used hereinrefers to organic molecules which exert biological effects in vivo.Examples of active agents include, without limitation, enzymes, receptorantagonists or agonists, hormones, growth factors, autogenous bonemarrow, antibiotics, antimicrobial agents and antibodies. The term“active agent” is also intended to encompass various cell types andgenes which can be incorporated into the compositions of the invention.The term “active agent” is also intended to encompass combinations ormixtures of two or more active agents, as defined above.

Preferred active agents for use in the compositions of the presentinvention include growth factors, such as transforming growth factors(TGFs), fibroblast growth factors (FGFs), platelet derived growthfactors (PDGFs), epidermal growth factors (EGFs), connective tissueactivated peptides (CTAPs), osteogenic factors, and biologically activeanalogs, fragments, and derivatives of such growth factors. Members ofthe transforming growth factor (TGF) supergene family, which aremultifunctional regulatory proteins, are particularly preferred. Membersof the TGF supergene family include the beta transforming growth factors(for example, TGF-β1, TGF-β2, TGF-β3); bone morphogenetic proteins (forexample, BMP-1, BMP-2, BMP-3, BMP-4, BMP-5, BMP-6, BMP-7, BMP-8, BMP-9);heparin-binding growth factors (for example, fibroblast growth factor(FGF), epidermal growth factor (EGF), platelet-derived growth factor(PDGF), insulin-like growth factor (IGF)); Inhibins (for example,Inhibin A, Inhibin B); growth differentiating factors (for example,GDF-1); and Activins (for example, Activin A, Activin B, Activin AB).

Growth factors can be isolated from native or natural sources, such asfrom mammalian cells, or can be prepared synthetically, such as byrecombinant DNA techniques or by various chemical processes. Inaddition, analogs, fragments, or derivatives of these factors can beused, provided that they exhibit at least some of the biologicalactivity of the native molecule. For example, analogs can be prepared byexpression of genes altered by site-specific mutagenesis or othergenetic engineering techniques.

Biologically active agents may be incorporated into the crosslinkedsynthetic polymer composition by admixture. Alternatively, the agentsmay be incorporated into the crosslinked polymer matrix, as describedabove, by binding these agents with the functional groups on thesynthetic polymers. Processes for covalently binding biologically activeagents such as growth factors using functionally activated polyethyleneglycols are described in commonly assigned U.S. Pat. No. 5,162,430,issued Nov. 10, 1992, to Rhee et al. Such compositions preferablyinclude linkages that can be easily biodegraded, for example as a resultof enzymatic degradation, resulting in the release of the active agentinto the target tissue, where it will exert its desired therapeuticeffect.

A simple method for incorporating biologically active agents containingnucleophilic groups into the crosslinked polymer composition involvesmixing the active agent with the first synthetic polymer (or firstsynthetic polymer/collagen mixture) prior to adding the second syntheticpolymer. This procedure will result in covalent binding of the activeagent to the crosslinked polymer composition, producing a highlyeffective sustained release composition.

The type and amount of active agent used will depend, among otherfactors, on the particular site and condition to be treated and thebiological activity and pharmacokinetics of the active agent selected.

Use of Crosslinked Synthetic Polymers to Deliver Cells or Genes

The crosslinked polymer compositions of the present invention can alsobe used to deliver various types of living cells or genes to a desiredsite of administration in order to form new tissue. The term “genes” asused herein is intended to encompass genetic material from naturalsources, synthetic nucleic acids, DNA, antisense-DNA and RNA.

When used to deliver cells, for example, mesenchymal stem cells can bedelivered to produce cells of the same type as the tissue into whichthey are delivered. Mesenchymal stem cells are not differentiated andtherefore can differentiate to form various types of new cells due tothe presence of an active agent or the effects (chemical, physical,etc.) of the local tissue environment. Examples of mesenchymal stemcells include osteoblasts, chondrocytes, and fibroblasts. Osteoblastscan be delivered to the site of a bone defect to produce new bone;chondrocytes can be delivered to the site of a cartilage defect toproduce new cartilage; fibroblasts can be delivered to produce collagenwherever new connective tissue is needed; neurectodermal cells can bedelivered to form new nerve tissue; epithelial cells can be delivered toform new epithelial tissues, such as liver, pancreas, etc.

The cells or genes may be either allogeneic or xenogeneic in origin. Forexample, the compositions can be used to deliver cells or genesfromother species which have been genetically modified. Because thecompositions of the invention are not easily degraded in vivo, cells andgenes entrapped within the crosslinked polymer compositions will beisolated from the patient's own cells and, as such, will not provoke animmune response in the patient. In order to entrap the cells or geneswithin a crosslinked polymer matrix, the first polymer and the cells orgenes may be pre-mixed, then the second polymer is mixed into the firstpolymer/cell or gene mixture to form a crosslinked matrix, therebyentrapping the cells or genes within the matrix.

As discussed above for biologically active agents, when used to delivercells or genes, the synthetic polymers preferably also containbiodegradable groups to aid in controlled release of the cells or genesat the intended site of delivery.

Use of the Crosslinked Synthetic Polymers as Bioadhesives

We have found that the preferred compositions of the invention tend tohave unusually high tackiness, making them particularly suitable for useas bioadhesives, for example, for use in surgery. As used herein, theterms “bioadhesive”, “biological adhesive”, and “surgical adhesive” areused interchangeably to refer to biocompatible compositions capable ofeffecting temporary or permanent attachment between the surfaces of twonative tissues, or between a native tissue surface and a non-nativetissue surface or a surface of a synthetic implant.

In a general method for effecting the attachment of a first surface to asecond surface, the first synthetic polymer and the second syntheticpolymer are applied to a first surface, then the first surface iscontacted with a second surface to effect adhesion between the firstsurface and the second surface. Preferably, the first synthetic polymerand second synthetic polymer are first mixed to initiate crosslinking,then delivered to a first surface before substantial crosslinking hasoccurred between the nucleophilic groups on the first synthetic polymerand the electrophilic groups on the second synthetic polymer. The firstsurface is then contacted with the second surface, preferablyimmediately, to effect adhesion between the two surfaces. At least oneof the first and second surfaces is preferably a native tissue surface.

For example, the first synthetic polymer and second synthetic polymerare generally provided in separate syringes, the contents of which arethen mixed together using syringe-to-syringe mixing techniques justprior to delivery to a first surface. The first synthetic polymer andsecond synthetic polymer are preferably mixed for a minimum of 20(preferably, 20 to 100; more preferably, 30 to 60) passes to ensureadequate mixing. As crosslinking between the corresponding reactivegroups on the two synthetic polymers is generally initiated during themixing process, it is important to deliver the reaction mixture to thefirst surface as soon as possible after mixing.

The reaction mixture can be extruded onto the first surface from theopening of a syringe or other appropriate extrusion device. Followingapplication, the extruded reaction mixture can be spread over the firstsurface using a spatula, if necessary. Alternatively, the firstsynthetic polymer and the second synthetic polymer can be mixed togetherin an appropriate mixing dish or vessel, then applied to the firstsurface using a spatula.

In another method for preparing the reaction mixture, the firstsynthetic polymer and second synthetic polymer are contained in separatechambers of a spray can or bottle with a nozzle, or other appropriatespraying device. In this scenario, the first and second polymers do notactually mix until they are expelled together from the nozzle of thespraying device. Following application of the reaction mixture to asurface containing collagen, the first surface is contacted with asecond surface. If the two surfaces are contacted before substantialcrosslinking has occurred between the synthetic polymer and thecrosslinking agent, the reactive groups on the crosslinking agent willalso covalently bond with primary amino groups on lysine residues ofcollagen molecules present on either or both of the surfaces, providingimproved adhesion.

The two surfaces may be held together manually, or using otherappropriate means, while the crosslinking reaction is proceeding tocompletion. Crosslinking is typically complete within 5 to 60 minutesafter mixing of the first and second synthetic polymers. However, thetime required for complete crosslinking to occur is dependent on anumber of factors, including the types and molecular weights of the twosynthetic polymers and, most particularly, the concentrations of the twosynthetic polymers (i.e., higher concentrations result in fastercrosslinking times).

At least one of the first and second surfaces is preferably a nativetissue surface. As used herein, the term “native tissue” refers tobiological tissues that are native to the body of the specific patientbeing treated. As used herein, the term “native tissue” is intended toinclude biological tissues that have been elevated or removed from onepart of the body of a patient for implantation to another part of thebody of the same patient (such as bone autografts, skin flap autografts,etc.). For example, the compositions of the invention can be used toadhere a piece of skin from one part of a patient's body to another partof the body, as in the case of a burn victim.

The other surface may be a native tissue surface, a non-native tissuesurface, or a surface of a synthetic implant. As used herein, the term“non-native tissue” refers to biological tissues that have been removedfrom the body of a donor patient (who may be of the same species or of adifferent species than the recipient patient) for implantation into thebody of a recipient patient (e.g., tissue and organ transplants). Forexample, the crosslinked polymer compositions of the present inventioncan be used to adhere a donor cornea to the eye of a recipient patient.

As used herein, the term “synthetic implant” refers to any biocompatiblematerial intended for implantation into the body of a patient notencompassed by the above definitions for native tissue and non-nativetissue. Synthetic implants include, for example, artificial bloodvessels, heart valves, artificial organs, bone prostheses, implantablelenticules, vascular grafts, stents, and stent/graft combinations, etc.

Use of Crosslinked Synthetic Polymers in Ophthalmic Applications

Because of their optical clarity, the crosslinked polymer compositionsof the invention which do not contain collagen are particularly wellsuited for use in ophthalmic applications. For example, a syntheticlenticule for correction of vision can be attached to the Bowman's layerof the cornea of a patient's eye using the methods of the presentinvention. As disclosed in commonly assigned, allowed U.S. applicationSer. No. 08/147,227, filed Nov. 3, 1993, by Rhee et al., a chemicallymodified collagen (such as succinylated or methylated collagen) which isin substantially nonfibrillar form at pH 7 can be crosslinked using asynthetic hydrophilic polymer, then molded into a desired lenticularshape and allowed to complete crosslinking. The resulting crosslinkedcollagen lenticule can then be attached to the Bowman's layer of ade-epithelialized cornea of a patient's eye using the methods of thepresent invention. By applying the reaction mixture comprising the firstand second synthetic polymers to the anterior surface of the cornea,then contacting the anterior surface of the cornea with the posteriorsurface of the lenticule before substantial crosslinking has occurred,electrophilic groups on the second synthetic polymer will alsocovalently bind with collagen molecules in both the corneal tissue andthe lenticule to firmly anchor the lenticule in place. (Alternatively,the reaction mixture can be applied first to the posterior surface ofthe lenticule, which is then contacted with the anterior surface of thecornea.)

The compositions of the present invention are also suitable for use invitreous replacement.

Use of Crosslinked Synthetic Polymer Compositions in Tissue Augmentation

The crosslinked polymer compositions of the invention can also be usedfor augmentation of soft or hard tissue within the body of a mammaliansubject. As such, they may be better than currently marketedcollagen-based materials product for soft tissue augmentation, becausethey are less immunogenic and more persistent. Examples of soft tissueaugmentation applications include sphincter (e.g., urinary, anal,esophageal) sphincter augmentation and the treatment of rhytids andscars. Examples of hard tissue augmentation applications include therepair and/or replacement of bone and/or cartilaginous tissue.

The compositions of the invention are particularly suited for use as areplacement material for synovial fluid in osteoarthritic joints, wherethe crosslinked polymer compositions serve to reduce joint pain andimprove joint function by restoring a soft hydrogel network in thejoint. The crosslinked polymer compositions can also be used as areplacement material for the nucleus pulposus of a damagedintervertebral disk. As such, the nucleus pulposus of the damaged diskis first removed, then the crosslinked polymer composition is injectedor otherwise introduced into the center of the disk. The composition mayeither be crosslinked prior to introduction into the disk, or allowed tocrosslink in situ.

In a general method for effecting augmentation of tissue within the bodyof a mammalian subject, the first and second synthetic polymers areinjected simultaneously to a tissue site in need of augmentation througha small-gauge (e.g., 25-32 gauge) needle. Once inside the patient'sbody, the nucleophilic groups on the first synthetic polymer and theelectrophilic groups on the second synthetic polymer will react witheach other to form a crosslinked polymer network in situ. Electrophilicgroups on the second synthetic polymer may also react with primary aminogroups on lysine residues of collagen molecules within the patient's owntissue, providing for “biological anchoring” of the compositions withthe host tissue.

Use of the Crosslinked Synthetic Polymer Compositions to PreventAdhesions

Another use of the crosslinked polymer compositions of the invention isto coat tissues in order to prevent the formation of adhesions followingsurgery or injury to internal tissues or organs. In a general method forcoating tissues to prevent the formation of adhesions following surgery,the first and second synthetic polymers are mixed, then a thin layer ofthe reaction mixture is applied to the tissues comprising, surrounding,and/or adjacent to the surgical site before substantial crosslinking hasoccurred between the nucleophilic groups on the first synthetic polymerand the electrophilic groups on the second synthetic polymer.Application of the reaction mixture to the tissue site may be byextrusion, brushing, spraying (as described above), or by any otherconvenient means.

Following application of the reaction mixture to the surgical site,crosslinking is allowed to continue in situ prior to closure of thesurgical incision. Once crosslinking has reached equilibrium, tissueswhich are brought into contact with the coated tissues will not stick tothe coated tissues. At this point in time, the surgical site can beclosed using conventional means (sutures, etc.).

In general, compositions that achieve complete crosslinking within arelatively short period of time (i.e., 5-15 minutes following mixture ofthe first synthetic polymer and the second synthetic polymer) arepreferred for use in the prevention of surgical adhesions, so that thesurgical site may be closed relatively soon after completion of thesurgical procedure.

Use of the Crosslinked Synthetic Polymers to Coat Implants

Another use of the crosslinked polymer compositions of the invention isas a coating material for synthetic implants. In a general method forcoating a surface of a synthetic implant, the first and second syntheticpolymers are mixed, then a thin layer of the reaction mixture is appliedto a surface of the implant before substantial crosslinking has occurredbetween the nucleophilic groups on the first synthetic polymer and theelectrophilic groups on the second synthetic polymer. In order tominimize cellular and fibrous reaction to the coated implant, thereaction mixture is preferably prepared to have a net neutral charge.Application of the reaction mixture to the implant surface may be byextrusion, brushing, spraying (as described above), or by any otherconvenient means. Following application of the reaction mixture to theimplant surface, crosslinking is allowed to continue until completecrosslinking has been achieved.

Although this method can be used to coat the surface of any type ofsynthetic implant, it is particularly useful for implants where reducedthrombogenicity is an important consideration, such as artificial bloodvessels and heart valves, vascular grafts, vascular stents, andstent/graft combinations. The method may also be used to coatimplantable surgical membranes (e.g., monofilament polypropylene) ormeshes (e.g., for use in hernia repair). Breast implants may also becoated using the above method in order to minimize capsular contracture.

The compositions of the present invention may also be used to coatlenticules, which are made from either naturally occurring or syntheticpolymers.

Use of the Crosslinked Synthetic Polymers to Treat Aneurism

The crosslinked polymer compositions of the invention can be extruded ormolded in the shape of a string or coil, then dehydrated. The resultingdehydrated string or coil can be delivered via catheter to the site of avascular malformation, such as an aneurysm, for the purpose of vascularocclusion and, ultimately, repair of the malformation. The dehydratedstring or coil can be delivered in a compact size and will rehydrateinside the blood vessel, swelling several times in size compared to itsdehydrated state, while maintaining its original shape.

Other Uses for the Crosslinked Synthetic Polymers

As discussed in commonly assigned, copending application Ser. No.08/574,050, filed Dec. 18, 1995, which is incorporated herein byreference, the crosslinked polymer compositions of the invention can beused to block or fill various lumens and voids in the body of amammalian subject. The compositions can also be used as biosealants toseal fissures or crevices within a tissue or structure (such as avessel), or junctures between adjacent tissues or structures, to preventleakage of blood or other biological fluids.

The crosslinked polymer compositions can also be used as a largespace-filling device for organ displacement in a body cavity duringsurgical or radiation procedures, for example, to protect the intestinesduring a planned course of radiation to the pelvis.

The crosslinked polymer compositions of the invention can also be coatedonto the interior surface of a physiological lumen, such as a bloodvessel or Fallopian tube, thereby serving as a sealant to preventrestenosis of the lumen following medical treatment, such as, forexample, balloon catheterization to remove arterial plaque deposits fromthe interior surface of a blood vessel, or removal of scar tissue orendometrial tissue from the interior of a Fallopian tube. A thin layerof the reaction mixture is preferably applied to the interior surface ofthe vessel (for example, via catheter) immediately following mixing ofthe first and second synthetic polymers. Because the compositions of theinvention are not readily degradable in vivo, the potential forrestenosis due to degradation of the coating is minimized. The use ofcrosslinked polymer compositions having a net neutral charge furtherminimizes the potential for restenosis.

EXAMPLES

The following examples are put forth so as to provide those of ordinaryskill in the art with a complete disclosure and description of how tomake the preferred embodiments of the conjugates, compositions, anddevices and are not intended to limit the scope of what the inventorsregard as their invention. Efforts have been made to ensure accuracywith respect to numbers used (e.g., amounts, temperature, molecularweight, etc.) but some experimental errors and deviation should beaccounted for. Unless indicated otherwise, parts are parts by weight,molecular weight is weight average molecular weight, temperature is indegrees Centigrade, and pressure is at or near atmospheric.

Example 1 Preparation of Crosslinked Multi-Amino PEG Compositions

0.15 grams of di-amino PEG (3400 MW, obtained from Shearwater Polymers,Huntsville, Ala.) in 250 ul of water was mixed with 0.1 g oftrifunctionally activated SC-PEG (5000 MW, also obtained from ShearwaterPolymers) using syringe-to-syringe mixing. The reaction mixture wasextruded onto a petri dish and formed a soft gel at room temperature.

0.15 gram of di-amino PEG in 250 μl of water was mixed with 0.1 g oftetrafunctionally activated SE-PEG (also from Shearwater Polymers) usingsyringe-to-syringe mixing. The reaction mixture was extruded onto apetri dish and formed a soft gel at room temperature.

Example 2 Preparation of Crosslinked Multi-Amino PEG Compositions

The following stock solutions of various di-amino PEGs were prepared:

Ten (10) grams of Jeffamine ED-2001 (obtained from Texaco ChemicalCompany, Houston, Tex.) was dissolved in 9 ml of water.

Ten (10) grams of Jeffamine ED-4000 (also obtained from Texaco ChemicalCompany) was dissolved in 9 ml of water.

0.1 grams of di-amino PEG (3400 MW, obtained from Shearwater Polymers,Huntsville, Ala.) was dissolved in 300 μl of water.

Each of the three di-amino PEG solutions prepared above was mixed withaqueous solutions of trifunctionally activated SC-PEG (TSC-PEG, 5000 MW,also obtained from Shearwater Polymers) as set forth in Table 1, below.TABLE 1 Preparation of Crosslinked Polymer Compositions Di-amino PEGTSC-PEG + Aqueous Solvent  50 ul  0 mg + 50 μl water  50 ul 10 mg + 50μl PBS  50 ul 10 mg + 100 μl PBS 250 ul 50 mg + 500 μl PBS

The solutions of di-amino PEG and TSC-PEG were mixed usingsyringe-to-syringe mixing. Each of the materials was extruded from thesyringe and allowed to set for 1 hour at 37° C. Each of the materialsformed a gel. In general, the gels became softer with increasing watercontent; the gels containing the least amount of aqueous solvent (wateror PBS) were firmest.

Example 3 Characterization of Crosslinked Multi-Amino PEG Compositions

Fifty (50) milligrams of tetra-amino PEG (10,000 MW, obtained fromShearwater Polymers, Huntsville, Ala.) in 0.5 ml PBS was mixed, usingsyringe-to-syringe mixing, with 50 mg of tetrafunctionally activatedSE-PEG (“tetra SE-PEG”, 10,000 MW, also obtained from ShearwaterPolymers) in 0.5 ml PBS or trifunctionally activated SC-PEG (“triSC-PEG”, 5000 MW, also obtained from Shearwater Polymers) in 0.5 ml PBS.

Syringes containing each of the two mixtures were incubated at 37° C.for approximately 16 hours. Both compositions formed elastic gels. Thegels were pushed out of the syringes and sliced into 5-6 mm thick diskshaving a diameter of 5 mm, for use in compression and swellabilitytesting, as described below.

Compression force versus displacement for the two gels was measured inthe Instron Universal Tester, Model 4202, at a compression rate of 2 mmper minute, using disks of the two gels prepared as described above.Compression force (in Newtons) versus gel displacement (in millimeters)is shown in FIGS. 1 and 2 for gels prepared using the tetra SE-PEG andtri SC-PEG, respectively.

Under compression forces as high as 30-35 Newtons, the gels did notbreak, but remained elastic.

Disks of each of the two gels, prepared as described above, were weighedand the dimensions (diameter and length) measured. The disks were thenimmersed in PBS and incubated at 37° C. After 3 days incubation, thedisks were removed from the PBS, weighed, and measured. Results ofswellability testing are shown in Table 2, below. TABLE 2 SwellabilityTesting of Crosslinked Multi-amino PEG Compositions Dimensions (in mm)Gel Weight (in grams) (diameter/thickness) Crosslinking Before AfterBefore After Agent Swelling Swelling Swelling Swelling Tetra SE-PEG0.116 0.310 5.0/5.0 7.1/8.1 Tri SC-PEG 0.131 0.287 5.0/6.0 6.4/8.5

As shown above, the gels swelled two to three times in weight, as wellas swelling an average of about 50% in both diameter and thickness.

Example 4 Preparation of Crosslinked PolyLysine Compositions

Ten (10) milligrams of poly-L-lysine hydrobromide (8,000 MW, obtainedfrom Peninsula Laboratories, Belmont, Calif.) in 0.1 ml phosphate buffer(0.2 M, pH=6.6) was mixed with 10 mg of tetrafunctionally activatedSE-PEG (10,000 MW, obtained from Shearwater Polymers, Huntsville, Ala.)in 0.1 ml PBS. The composition formed a soft gel almost immediately.

Example 5 Preparation and Mechanical Testing of Crosslinked Multi-AminoPEG Compositions

Gels comprising tetra-amino PEG (10,000 MW, obtained from ShearwaterPolymers, Huntsville, Ala.) and 1-4% (by weight) of tetrafunctionallyactivated SE-PEG (“tetra SE-PEG”, 10,000 MW, also obtained fromShearwater Polymers) were prepared by mixing the tetra-amino PEG (at aconcentration of 25 mg/ml in water) with the tetra SE-PEG (in PBS) in apetri dish. The resulting tetra-amino PEG/SE-PEG mixtures were incubatedfor 16 hours at 37° C.

The mixture containing 1% SE-PEG did not form a gel due to the lowSE-PEG concentration. The mixture containing 2% SE-PEG formed a gel atsome point during the 16-hour incubation period. The mixtures containing3 and 4% SE-PEG formed gels within approximately 4-6 minutes of mixing.The gel containing 2% SE-PEG was readily extrudable through a 30-gaugeneedle; the gel containing 3% SE-PEG could be extruded through a27-gauge needle.

The effect of elevated temperature on gel formation was evaluated. Gelscomprising tetra-amino PEG and 2.5% (by weight) tetra SE-PEG wereprepared and incubated at temperatures of 37° C. and 40-50° C. Elevatedtemperature was found to have a marked effect on gelation time: thetetra-amino PEG/SE-PEG mixture incubated at 37° C. formed a gel withinapproximately 20-25 minutes, whereas mixtures incubated at 40-50° C.formed gels within approximately 5 minutes. Both gels were extrudablethrough a 27-gauge needle.

The effect of pH on gel formation was evaluated. Gels comprisingtetra-amino PEG and 2.5% (by weight) tetra SE-PEG were prepared as setforth in Table 3, below. TABLE 3 Effect of pH on Gel Formation ofTetra-amino PEG/Tetra SE-PEG Formulations pH of pH of Tetra-amino pH ofResulting Gelation PEG Tetra SE-PEG Mixture Gelation Time Temp. 10 4.16.9 10-15 minutes 45° C. 10 7.0 7.2   <5 minutes 45° C.

Extrudability through a 27-gauge needle was evaluated for gelscomprising tetra-amino PEG and 1-3% (by weight) tetra SE-PEG. The gelswere contained within 1-cc syringes. The force required to depress thesyringe plunger at a rate of 5 centimeters per minute was measured usingthe Instron Universal Tester, Model 4202. Results of extrusion testingare presented in Table 4, below. TABLE 4 Extrusion of Tetra-aminoPEG/Tetra SE-PEG Gels Through a 27-Gauge Needle Concentration of SE-PEG(by weight) Extrusion Force (N) 1.5-2% 10-11 2-2.5% 52 2.5-3% 88

Extrusion forces of 100 N or less are considered acceptable for manualinjection without the aid of a syringe assist device.

Tensile strength (i.e., elasticity) of 3 mm thick gels comprisingtetra-amino PEG and 2.5, 5, and 10% (by weight) tetra SE-PEG wasmeasured using the Instron Universal Tester, Model 4202. Gels of varyinginitial lengths were stretched at a rate of 10 millimeters per minute.Length of each gel, strain at failure (change in length as a percentageof the initial length), and force at failure are set forth in Table 5,below. TABLE 5 Tensile Strength of Tetra-amino PEG/Tetra SE-PEG GelsSE-PEG Conc. Initial Strain at Force at (wt %) Length (cm) FailureFailure (N) 10 1.4 139% 0.4 10 1.9 99% 0.5 10 2.5 78% 0.5 5 1.3 111% 0.25 1.3 99% 0.2 5 1.6 94% 0.2 2.5 1.0 237% <0.1 2.5 1.5 187% <0.1 2.5 1.7129% <0.1

Gels containing 5 and 10% tetra SE-PEG approximately doubled in lengthprior to breaking. Gels containing 2.5% SE-PEG approximately tripled inlength prior to breaking, but were considerably weaker (i.e., lowerforce at failure) than the more highly crosslinked gels.

Example 6 Effect of pH on Gel Formation of Tetra-Amino PEG/Tetra SE-PEGFormulations

Gels comprising various concentrations of tetra-amino PEG and tetraSE-PEG at pH 6, 7, and 8 were prepared in petri dishes. Following mixingof the tetra-amino PEG and tetra SE-PEG, the dishes were tiltedrepeatedly; the gelation time was considered to be the point at whichthe formulation ceased to flow. The effect of pH on gelation time of thevarious tetra-amino PEG/tetra SE-PEG formulations at room temperature isshown in Table 6, below. TABLE 6 Effect of pH on Gel Formation ofTetra-amino PEG/Tetra SE-PEG Formulations Tetra-amino PEG Tetra SE-PEGConc. (mg/ml) Conc. (mg/ml) pH Gelation Time  20  20 6 >90.0 min 20 20 7  20.0 min 20 20 8    1.4 min 50 50 6   24.0 min 50 50 7    3.5 min 5050 8   10.0 sec 100 100 6    9.0 min 100 100 7   47.0 sec 100 100 8  10.0 sec 200 200 6    2.0 min 200 200 7    9.0 sec 200 200 8    5.0sec

The time required for gel formation decreased with increasing pH andincreasing tetra-amino PEG and tetra SE-PEG concentrations.

Example 7 Culturing of Cells in Crosslinked Multi-Amino PEG Matrix

Thirty (30) milligrams of tetra-amino PEG (10,000 MW, obtained fromShearwater Polymers, Huntsville, Ala.) was dissolved in 0.6 ml PBS, thensterile filtered. Thirty (30) milligrams of tetrafunctionally activatedSE-PEG (“tetra SE-PEG”, 10,000 MW, also obtained from ShearwaterPolymers) was dissolved in 0.6 ml of PBS, then sterile filtered.

The solutions of tetra-amino PEG and tetra SE-PEG were mixed togetherwith a pellet containing human skin fibroblast (“HSF”) cells (CRL #1885,passage 4, obtained from American Tissue Type Culture Collection,Rockville, Md.). Two hundred fifty (250) microliters of the resultingcell-containing tetra-amino PEG/tetra SE-PEG (PEG-PEG) solution wasdispensed into each of two wells on a 48-well culture plate and allowedto gel for approximately 5 minutes at room temperature. One (1) milliterof Dulbecco Modified Eagle's Media (supplemented with 10% fetal bovineserum, L-glutamine, penicillin-streptomycin, and non-essential aminoacids) was added to each of the two wells. The concentration of cellswas approximately 3×10⁵ cells per milliliter of tetra-amino PEG/tetraSE-PEG solution, or 7.5×10⁵ cells per well.

To prepare a control, a pellet of HSF cells were suspended in 1.2 ml ofcomplete media. Two hundred fifty (250) microliters of the controlmixture was dispensed into each of three wells on the same 48-wellculture plate as used above. Each well was estimated to containapproximately 7.5×10⁵ cells. Each well was given fresh media every otherday.

Initially, the cell-containing tetra-amino PEG/tetra SE-PEG gels wereclear and the cells were found to be densely populated and spheroidal inmorphology, indicating that there was little adhesion between the cellsand the PEG/PEG gel (the cells would normally assume a flattened,spindle-shaped morphology when adhered to a substrate, such as to thetreated plastic of the tissue culture plates). After three 3 daysincubation at 37° C., the media in the wells containing the PEG/PEG gelswas found to have lightened in color (Dulbecco Modified Eagle's Media isnormally red in color), indicating a pH change in the media Thisindicated that the cells were alive and feeding. At 7 days incubation at37° C., the cells were still spheroidal in morphology (indicating lackof adhesion to the gel) and the media had lightened even further,indicating that the cells were still viable and continued to feed.

On day 7, the contents of each well were placed in a 10% formalinsolution for histological evaluation. According to histologicalevaluation, an estimated 75% of the cells in the wells containing thePEG PEG gels appeared to be alive, but did not appear to be reproducing.

The results of the experiment indicate that HSF cells are viable in thetetra-amino PEG/tetra SE-PEG crosslinked gels, but did not seem toadhere to the gel and did not appear to reproduce while entrapped withinthe gel matrix. As described above, adherence or non-adherence of cellsto a substrate material can influence the cells' morphology. In certaintypes of cells, cellular morphology can, in turn, influence certaincellular functions. Therefore, non-adherence of the cells to the PEG-PEGgel matrix may be an advantage in the delivery of particular cell typeswhose function is influenced by cell morphology. For example, theability of cartilage cells to produce extracellular matrix materials isinfluenced by cellular morphology: when the cells are in the flattened,spindle-shaped configuration, the cells are in reproductive mode; whenthe cells are in the spheroidal configuration, reproduction stops, andthe cells begin to produce extracellular matrix components.

Because the PEG-PEG gels are not readily degraded in vivo, the gels maybe particularly useful in cell delivery applications where it isdesirable that the cells remain entrapped within the matrix for extendedperiods of time:

1. A method for preparing a biocompatible crosslinked composition,comprising admixing, under crosslinking conditions, (a) a firstcrosslinkable component having m nucleophilic groups, wherein m≧2, with(b) a second crosslinkable component having n electrophilic groupscapable of reaction with the m nucleophilic groups to form covalentbonds, wherein n≧2 and m+n≧5, wherein each of the first and secondcrosslinkable components is biocompatible and synthetic, andcrosslinking of the components results in the biocompatible crosslinkedcomposition.
 2. The method of claim 1, wherein the m nucleophilic groupsin the first crosslinkable component are identical.
 3. The method ofclaim 1, wherein at least two of the m nucleophilic groups in the firstcrosslinkable component are different.
 4. The method of claim 1, whereinthe n electrophilic groups in the second crosslinkable component areidentical.
 5. The method of claim 2, wherein the n electrophilic groupsin the second crosslinkable component are identical.
 6. The method ofclaim 3, wherein the n electrophilic groups in the second crosslinkablecomponent are identical.
 7. The method of claim 1, wherein at least twoof the n electrophilic groups in the second crosslinkable component aredifferent.
 8. The method of claim 2, wherein at least two of the nelectrophilic groups in the second crosslinkable component aredifferent.
 9. The method of claim 3, wherein at least two of the nelectrophilic groups in the second crosslinkable component aredifferent.
 10. The method of claim 1, wherein the m nucleophilic groupsare bound to the first crosslinkable component through linking groups.11. The method of claim 1, wherein the n electrophilic groups are boundto the second crosslinkable component through linking groups.
 12. Themethod of claim 1, wherein at least one of the first and secondcrosslinkable components is comprised of a hydrophilic polymer.
 13. Themethod of claim 1, wherein at least one of the first and secondcrosslinkable components is comprised of a hydrophobic polymer.
 14. Themethod of claim 1, wherein the m nucleophilic groups are primary aminogroups.
 15. The method of claim 14, wherein the first crosslinkablecomponent is C₂-C₆ hydrocarbyl substituted with amino groups.
 16. Themethod of claim 14, wherein the first crosslinkable component is asecondary or tertiary amine NR₁R₂R₃ wherein R₁ is hydrogen or anamino-substituted lower alkyl group, and R₂ and R₃ are amino-substitutedlower alkyl groups.
 17. The method of claim 14, wherein the nelectrophilic groups are selected from the group consisting ofsuccinimidyl ester, sulfosuccinimidyl ester, maleimido, epoxy,isocyanato, thioisocyanato, and ethenesulfonyl.
 18. The method of claim17, wherein the n electrophilic groups are selected from the groupconsisting of succinimidyl ester and sulfosuccinimidyl ester.
 19. Themethod of claim 1, wherein the m nucleophilic groups are sulfhydrylgroups.
 20. The method of claim 19, wherein the n electrophilic groupsare sulfhydryl-reactive groups selected so as to form a thioester,thioether, or disulfide linkage upon reaction with the sulfhydrylgroups.
 21. The method of claim 1, wherein n=2.
 22. The method of claim1, wherein m=2.
 23. The method of claim 1, wherein the crosslinkingconditions comprise admixture in an aqueous medium.
 24. The method ofclaim 23, wherein the first and second crosslinkable components eachrepresent about 0.5 wt. % to about 20 wt. % of the composition formedupon admixture.
 25. The method of claim 23, wherein the crosslinkingconditions further comprise admixture at a pH in the range of 7 to 8.26. The method of claim 25, wherein the first and second crosslinkablecomponents are at concentrations of 20 mg/ml to 200 mg/ml of thecomposition formed upon admixture.
 27. The method of claim 1, whereinthe first crosslinkable component is in an aqueous solution, the secondcrosslinkable component is in dry, particulate form, and admixingcomprises combining the second crosslinkable component with the aqueoussolution of the first crosslinkable component.
 28. The method of claim27, the first and second crosslinkable components each represent about0.5 wt. % to about 20 wt. % of the composition formed upon admixture.29. The method of claim 27, wherein the crosslinking conditions furthercomprise admixture at a pH in the range of 7 to
 8. 30. The method ofclaim 29, wherein the first and second crosslinkable components are atconcentrations of 20 mg/ml to 200 mg/ml of the composition formed uponadmixture.
 31. The method of claim 1, wherein the first crosslinkablecomponent is present in a molar excess relative to the secondcrosslinkable component.
 32. The method of claim 1, wherein the secondcrosslinkable component is present in a molar excess relative to thefirst crosslinkable component.
 33. A biocompatible crosslinkedcomposition prepared by the process comprising admixing, undercrosslinking conditions, (a) a first crosslinkable component having mnucleophilic groups, wherein m≧2, with (b) a second crosslinkablecomponent having n electrophilic groups capable of reaction with the mnucleophilic groups to form covalent bonds, wherein n≧2 and m+n≧5,wherein each of the first and second crosslinkable components isbiocompatible and synthetic, and crosslinking of the components resultsin the biocompatible crosslinked composition.
 34. The composition ofclaim 33, wherein the m nucleophilic groups in the first crosslinkablecomponent are identical.
 35. The composition of claim 33, wherein atleast two of the m nucleophilic groups in the first crosslinkablecomponent are different.
 36. The composition of claim 33, wherein the nelectrophilic groups in the second crosslinkable component areidentical.
 37. The composition of claim 34, wherein the n electrophilicgroups in the second crosslinkable component are identical.
 38. Thecomposition of claim 35, wherein the n electrophilic groups in thesecond crosslinkable component are identical.
 39. The composition ofclaim 33, wherein at least two of the n electrophilic groups in thesecond crosslinkable component are different.
 40. The composition ofclaim 34, wherein at least two of the n electrophilic groups in thesecond crosslinkable component are different.
 41. The composition ofclaim 35, wherein at least two of the n electrophilic groups in thesecond crosslinkable component are different.
 42. The composition ofclaim 33, wherein the m nucleophilic groups are bound to the firstcrosslinkable component through linking groups.
 43. The composition ofclaim 33, wherein the n electrophilic groups are bound to the secondcrosslinkable component through linking groups.
 44. The composition ofclaim 33, wherein at least one of the first and second crosslinkablecomponents is comprised of a hydrophilic polymer.
 45. The composition ofclaim 33, wherein at least one of the first and second crosslinkablecomponents is comprised of a hydrophobic polymer.
 46. The composition ofclaim 33, wherein the m nucleophilic groups are primary amino groups.47. The composition of claim 46, wherein the first crosslinkablecomponent is C₂-C₆ hydrocarbyl substituted with amino groups.
 48. Thecomposition of claim 46, wherein the first crosslinkable component is asecondary or tertiary amine NR₁R₂R₃ wherein R₁ is hydrogen or anamino-substituted lower alkyl group, and R₂ and R₃ are amino-substitutedlower alkyl groups.
 49. The composition of claim 46, wherein the nelectrophilic groups are selected from the group consisting ofsuccinimidyl ester, sulfosuccinimidyl ester, maleimido, epoxy,isocyanato, thioisocyanato, and ethenesulfonyl.
 50. The composition ofclaim 49, wherein the n electrophilic groups are selected from the groupconsisting of succinimidyl ester and sulfosuccinimidyl ester.
 51. Thecomposition of claim 33, wherein the m nucleophilic groups aresulfhydryl groups.
 52. The composition of claim 51, wherein the nelectrophilic groups are sulfhydryl-reactive groups selected so as toform a thioester, thioether, or disulfide linkage upon reaction with thesulfhydryl groups.
 53. The composition of claim 33, wherein n=2.
 54. Thecomposition of claim 33, wherein m=2.
 55. The composition of claim 33,wherein the crosslinking conditions comprise admixture in an aqueousmedium.
 56. The composition of claim 55, wherein the first and secondcrosslinkable components each represent about 0.5 wt. % to about 20 wt.% of the composition formed upon admixture.
 57. The composition of claim55, wherein the crosslinking conditions further comprise admixture at apH in the range of 7 to
 8. 58. The composition of claim 57, wherein thefirst and second crosslinkable components are at concentrations of 20mg/ml to 200 mg/ml of the composition formed upon admixture.
 59. Thecomposition of claim 33, wherein the first crosslinkable component is inan aqueous solution, the second crosslinkable component is in dry,particulate form, and admixing comprises combining the secondcrosslinkable component with the aqueous solution of the firstcrosslinkable component.
 60. The composition of claim 59, the first andsecond crosslinkable components each represent about 0.5 wt. % to about20 wt. % of the composition formed upon admixture.
 61. The compositionof claim 59, wherein the crosslinking conditions further compriseadmixture at a pH in the range of 7 to
 8. 62. The composition of claim61, wherein the first and second crosslinkable components are atconcentrations of 20 mg/ml to 200 mg/ml of the composition formed uponadmixture.
 63. The composition of claim 33, wherein the firstcrosslinkable component is present in a molar excess relative to thesecond crosslinkable component.
 64. The composition of claim 33, whereinthe second crosslinkable component is present in a molar excess relativeto the first crosslinkable component.